Virtual high-throughput screening (vHTS) can be used in drug discovery to replace experimental HTS or to preselect a subset of compounds for screening to reduce costs and time. The two main approaches to vHTS are structure-based or ligand-based screening. Structure-based screening does not need any known active molecules, but does need either a crystal structure or good homology model of the target protein. Ligand-based screening, on the other hand, does not need any knowledge about the target protein but identifies common patterns and features of known active molecules by creating a 2D fingerprint or 3D pharmacophore.
Scientists at Imperial College London/Equinox Pharma have developed a new rule-based vHTS methodology (INDDEx) that exploits logic-based machine learning to enhance performance in ligand-based screening. INDDEx (Investigational Novel Drug Discovery by Example) is particularly good at identifying actives that are structurally distinct from the training set, making it useful for scaffold-hopping.
INDDEx learns easily interpretable qualitative logic rules from active ligands. These rules – in the form of ‘an active molecule requires fragment A and fragment B separated by a distance in Angstroms’ or ‘an active molecule requires the presence of fragment C’ or ‘an active molecule must NOT contain fragment D’ – give an insight into chemistry, relate molecular substructure to activity and can be used to guide the next steps of drug design chemistry. These qualitative rules are then weighted using Support Vector Machines (SVMs) to produce QSAR rules that can be used to generate novel in silico hits.
INDDEx has been shown to be a powerful new approach to virtual screening whose strength lies in learning topological descriptors of multiple active compounds although, when considering scaffold hopping in isolation, INDDEx performs well even when there are small numbers of active molecules to learn from. One very attractive feature of INDDEx is that the rules that are produced can be readily understood and used by medicinal chemists. The technology has been extensively validated and shown to outperform comparable approaches (J. Phys. Chem. B 2012, 116, 6732-6739). In a study between Equinox and Imperial on sirtuin 2 (an NAD-dependent histone deacetylase) , INDDEx combined with structure-based docking was able to learn from only eight actives and identify a chemically novel hit that was experimentally validated to have an IC50 of 0.6 µM.
INDDEx has wide-scale applications including rescuing failed programmes, directing hit-to-lead programmes and scaffold-hopping. Furthermore, INDDEx has the potential to derive rules for off-target activities such as the hERG receptor.
If you would like to find out more about INDDEx and Equinox Pharma, please contact us.
With the rapidly growing body of biostructural information, structure-based drug design has increased in importance and a variety of computational methods have found a place in the drug discovery toolkit.
The de novo design program, SkelGen, was developed by De Novo Pharmaceuticals based on research begun in the Department of Pharmacology at the University of Cambridge. SkelGen constructs candidate ligands by assembling small molecular fragments within a protein target such as an enzyme or receptor (usually derived from X-ray crystal data). When growing a ligand, SkelGen uses information coded in the fragments and within its algorithm to favour synthetically tractable molecules. SkelGen is able to explore around one trillion low molecular weight, drug-like molecules using a default set of 1600 fragments. Since the accessible chemical space is so large, the majority of designed molecules are novel and patentable.
Whilst SkelGen can be run with minimal input, it also permits extensive control by the end-user, allowing the scientist to incorporate prior knowledge and insights into the drug design process. As well as completely de novo design, molecule generation can also be started from a user-defined fragment (for example, a low-affinity molecule identified by fragment-screening). SkelGen can also be used for scaffold hopping (chemotype switching) and focused library design.
Until recently SkelGen was only accessible through collaborations with De Novo Pharmaceuticals but is now available under both academic and commercial licenses. With these new licensing models, SkelGen can be a cost-effective (and accessible) tool for all scientists engaged in drug design. If you would like to find out more about SkelGen, please contact us.
Crystal structure of the ligand-binding domain of RORα complexed with cholesterol sulfate. PDB ID=1S0X
Scientists at the Scripps Research Institute
have reported on compounds that are able to suppress severity and disease progression in animal models of multiple sclerosis. The compounds, exemplified by SR1001, act by selectively suppressing a subset of T-helper cells characterised by their production of interleukin-17 (TH
17 cells). TH
17 cells have been implicated in a variety of autoimmune diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and systemic lupus erythematosus.
SR1001 selectively binds to two orphan nuclear receptors: retinoic acid receptor-related orphan receptors α and γt (RORα and RORγt). These receptors have indispensible roles in the development and function of TH17 cells, providing a mechanism for modulating one component of the immune system without general immunosuppression. The team reports that SR1001 induces a conformational change in the receptors that results in their reduced affinity for co-activators and increased affinity for co-repressors. The net result is inhibition of the receptors’ transcriptional activity.
SR1001 blocked the development of murine TH17 cells and inhibited cytokine production by differentiated murine and human TH17 cells. Although a drug is some way off, the team suggests that the results demonstrate the feasibility of targeting TH17 cells and the potential of such an approach for the treatment of autoimmune diseases.
The study is published in Nature.
Image: Flickr - Junichiro Aoyama
Bz-423, a mitochondrial F1
-ATP synthase inhibitor, that has previously shown promise for the treatment of autoimmune disorders such as lupus, arthritis and psoriasis has now been shown to halt the progression of established graft-versus-host disease (GVHD) in mouse models of allogeneic bone marrow transplantation. GVHD, in which functional immune cells in the transplanted marrow mount an immunological attack on the recipient, is a common complication of allogeneic bone marrow transplantation that can cause severe organ damage and even be life-threatening. As well as demonstrating that Bz-423 can reduce GVHD clinical scores and improve survival in mice, researchers at the University of Michigan
and the University of Florida have shed new light on the metabolism of alloreactive T cells.
Resting lymphocytes meet their minimal demand for ATP using low rates of oxidative phosphorylation. Upon activation, normal lymphocytes meet the increased demand for ATP by dramatically increasing their rate of aerobic glycolysis which also helps to maintain high levels of antioxidants in activated cells. Data from the present study suggest that alloreactive T cells, on the other hand, rely heavily on increased oxidative phosphorylation to generate more ATP. This difference in energy generation is also seen in pathogenic T cells involved in autoimmune diseases and provides a mechanistic basis for the specific elimination of pathogenic cells by Bz-423, whilst preserving normal immune function.
The study is published in Science Translational Medicine.
Lycera Corporation has a program to develop orally bioavailable F1F0-ATP synthase inhibitors that exploit bioenergetic abnormalities in pathologically activated lymphocytes and result in the selective silencing of these cells.
Image: Flickr - Janne Moren
Tool compounds are used to explore the role of a specific protein in a biological context and – it goes without saying – that to obtain meaningful results in a complex situation, the tool compound should have appropriate potency and selectivity. The family of phospholipase C (PLC) enzymes play important regulatory roles and a small molecule inhibitor, U73122, has been extensively used to provide evidence for the involvement of PLCs in many cellular pathways. Recent reports, however, have questioned the selectivity of U73122 and scientists at the University of North Carolina and GlaxoSmithKline have now discovered that, even in its interaction with PLCs, U73122 may not be quite what it seems. When the team explored the effects of U73122 on human PLCs in cell-free micellar systems, they found that the compound actually increased the enzymatic activity of a number of isoforms in a concentration- and time-dependent manner. At micromolar concentrations, U713122 increased the activity of PLCβ3 by up to eight-fold, that of PLCγ1 by more than ten-fold, and that of PLCβ2 by around two-fold; PLCδ1 was neither activated nor inhibited.
Activation of PLCβ3 was attenuated by competing nucleophiles, suggesting that activation involves covalent modification of the protein by the reactive maleimide group of U73122; the analogous succinimide, U73343, was not effective as an activator. Involvement of specific cysteine residues in the protein was demonstrated by LC/MS/MS experiments. Although N-ethyl maleimide (NEM) itself did not activate PLCβ3, excess NEM attenuated the U73122-mediated activation in a concentration-dependent manner. The authors propose an activation model in which U73122 irreversibly binds to multiple cysteine residues on PLCβ3 and acts as either a lipid anchor or interfacial recognition site for the enzyme, facilitating adsorption to the substrate interface (i.e. the micelle surface). The protein-linked U73122 increases the rate of lipase activity by keeping the enzyme in close proximity to substrate which is held in the membrane.
The study, which is published in the Journal of Biological Chemistry, provides strong evidence that U73122 activates PLC enzymes in cell-free systems, in contrast to its ‘established’ role as a specific inhibitor of this family. The authors suggest that U73122 may have opposing effects on cytosolic and membrane-bound enzymes and/or may modify other cellular nucleophiles and advise great caution when forming hypotheses based on the observed effects of U73122 in cellular systems.
Image: Flickr - Darren D
Multiple sclerosis (MS), believed to be an immune-mediated disorder, is the most common disabling condition of the central nervous system (CNS) affecting young adults. Infiltration of leukocytes into the brain, helped by upregulation of matrix metalloproteinases (MMPs) which cleave components of the extracellular matrix, plays a significant role in causing the demyelination and axonal degeneration associated with MS.
Synthesis of MMPs is regulated by extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), a multifunctional member of the immunoglobulin superfamily, and researchers at the Hotchkiss Brain Institute, University of Calgary have shown that EMMPRIN levels are significantly increased in the brain of MS patients, particularly in plaque-containing regions. The team also showed that EMMPRIN levels are increased in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EMMPRIN was upregulated on peripheral leukocytes before the appearance of MS-like symptoms and on infiltrating leukocytes and resident cells within the CNS once clinical symptoms had developed. Treating the mice with an anti-EMMPRIN antibody reduced MMP activity, infiltration of leukocytes into the CNS, and the severity of MS-like symptoms. To be effective, the antibody had to be administered at the onset of clinical signs, a time that is typically associated with significant infiltration of leukocytes into the CNS.
The study, which is published in the Journal of Neuroscience, suggests that blocking the activity of EMMPRIN may be a novel way to treat MS.
Image: Flickr - bearroast
The majority of Parkinson’s disease (PD) cases have no known cause, but have been associated with increased oxidative stress and mitochondrial dysfunction. Of the small proportion of hereditary cases, a number of defective genes have been identified including LRRK2 (PARK8), DJ-1 (PARK7), α-synuclein (SNCA) and parkin
(PARK2). Mutations in parkin
, which encodes an E3 ubiquitin ligase, are believed to interfere with the ability of parkin to clear the cell of its normal substrate proteins. Several substrates for parkin have been identified and shown to accumulate in the brain tissue of patients with hereditary PD.
Researchers at The University of Texas Health Science Center have now identified a link between the tyrosine kinase, c-Abl, and impaired parkin function. The scientists found that c-Abl was activated in cultured neuronal cells and the striatum of adult mice when subjected to oxidative and neuronal stress. They also identified parkin as a specific substrate for c-Abl and that the tyrosine-phosphorylated parkin lost its ubiquitin ligase activity.
The c-Abl inhibitor, imatinib (STI-571) was able to block the phosphorylation of parkin in vitro and in vivo, restoring ligase activity. Since there are several c-Abl inhibitors approved for the treatment of chronic myelogenous leukemia, tools are available to further explore the neuroprotective potential of c-Abl inhibition in sporadic PD.
The study is published in the Journal of Neuroscience.
Image: Flickr - Mohamed Hussain
Development of cancer is conventionally viewed as a gradual process, taking years to accumulate multiple point mutations and chromosomal rearrangements, and progressing through increasingly malignant phenotypes. New research by a team at the Wellcome Trust Sanger Institute
has shown that, in some cases, cancer can result from a single catastrophic event involving tens to hundreds of genomic rearrangements. Using advanced DNA sequencing techniques, the team found that 2-3% of cancer samples, across many common subtypes, had dramatic structural changes affecting highly localised regions of one or more chromosomes that could not be explained using standard models of DNA damage. This type of damage was especially common in bone cancers, where around 25% of samples showed signs of chromosomal crisis.
The team have proposed that such extensive damage is likely to occur when the chromosomes are condensed for mitosis and could be caused by ionising radiation or linked to telomere attrition. If the cell attempts to repair such extensive damage, the evidence suggests that the repair goes badly wrong, resulting in a genome that is riddled with mutations. In most cases, such haphazard repairs would be detrimental to the cell’s ability to survive and divide but in some cases can amplify cancer genes or inactivate tumour suppressor genes. It is remarkable that the cell can not only survive such a cataclysmic event but can emerge with a selective advantage.
The study is published in the journal, Cell.
Leukocytes: Friend or Foe to Transformed Cells? Image of a neutrophil (red) gliding over a doublet of transformed mucus-producing cells
Last week it was reported
that a daily (low) dose of aspirin can significantly reduce the risk of dying from a variety of cancers, and a study published in PloS Biology
now opens a new window on the role of inflammation in cancer.
Although the host immune system is known to have conflicting roles in cancer initiation and progression, both acting as a surveillance and elimination system but also assisting expansion and metastatic spread of tumours, very little is known about the very early role of the immune system in cancer. Using zebrafish larvae, researchers at the University of Bristol, the University of Manchester and the FIRC Institute of Molecular Oncology in Milan have now been able to observe, for the first time, how oncogene-transformed cells in the skin co-opt the innate immune system to promote their growth from the very earliest stages of development. The team exploited the translucency of the larvae to obtain live images of the earliest interactions between the cancer cells and the immune environment. Using larvae with fluorescently tagged leukocytes, the team were able to observe recruitment of neutrophils and macrophages to oncogene-transformed melanocytes or mucus-secreting cells. As well as engulfment of the transformed cells, the team saw many examples of cytoplasmic tethers linking the two cell types.
They discovered that a key attractant for the leukocytes was hydrogen peroxide. Both the transformed cells themselves and otherwise healthy neighbouring cells were found to produce hydrogen peroxide, which is also a key molecule that recruits neutrophils to a wound. Blocking the synthesis of hydrogen peroxide prevented recruitment of immune cells and reduced the number of transformed cells, suggesting that immune cells may provide trophic support to the transformed cells just as they promote repair at a site of tissue injury. Unlike the case of wound healing, however, where the inflammatory response resolves, the inflammatory response to transformed cells seems to amplify and progress towards a chronic inflammatory state similar to that seen in chronic non-healing wounds.
Image: Wikimedia Commons
Transient receptor potential (TRP) ion channels mediate a variety of sensations including thermal stimuli. The channels also react to multiple ligands: for example, TRPV1 channels respond to heat as well as to ligands which elicit a hot sensation such as capsaicin, whereas TRPM8 channels respond to cold and also to ligands which elicit a cool sensation such as menthol. Since TRP channels are expressed on sensory neurons, a number of groups are developing selective channel modulators for the treatment of pain.
Researchers led by a team at the Instituto de Neurociencias de Alicante have now shown that TRPM8 channels in the eye also regulate basal tear secretion. In studies in mice, the researchers found that cooling of the eye surface by 1-2°C by evaporation of the tear film can induce production of more tears by stimulation of TRPM8 in nerve endings in the cornea. In mice lacking TRPM8, the cornea was insensitive to cooling and the basal rate of tear production was greatly reduced. Tear production caused by exposure to irritants which is mediated by other channels such as TRPV1 was, however, unaffected in the TRPM8 deficient mice. In normal mice, tear secretion could also be decreased by raising the temperature of the cornea to 33-36°C. Tear production in humans is also regulated by cold – the basal rate of tear production is significantly lower at 43°C than at 18-20°C.
The study, which is published in the journal Nature Medicine, indicates that TRPM8 contributes to the regulation of basal tear flow and opens new possibilities for the treatment of dry eye syndrome by increasing tear secretion. Dry mucosal surfaces, including dry eye syndrome, are a common problem, particularly for the elderly with up to one third of people over the age of sixty five estimated to have dry eyes.